Download New Perspectives in Biological Nitrogen Fixation Research

New Perspectives in Biological Nitrogen Fixation Research
Qi Cheng
Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln,
Lincoln, Nebraska, NE 68588, USA
Tel: 402-403-0567; Fax: 402-472-7842; e-mail: [email protected]
Abstract
Nitrogen fixation, along with photosynthesis is the basis of all life on Earth. Current understanding
suggests that no plant fixes its own nitrogen. Some plants (mainly legumes) fix nitrogen via
symbiotic anaerobic microorganisms (mainly rhizobia). The nature of biological nitrogen fixation
is that the dinitrogenase catalyzes the reaction splitting triple bond inert atmospheric nitrogen (N 2)
into organic ammonia molecule (NH3). All known nitrogenases are found to be prokaryotic,
two-component complex and normally oxygen liable. The engineering of autonomous
nitrogen-fixing plants would not surprisingly be a long-term effort since it requires the assembly of
a complex enzyme and provision of anaerobic conditions. However, in the light of evolving protein
catalysts, the anaerobic enzyme has almost certainly been replaced in many reactions by the more
efficient and irreversible aerobic version that uses O2. On the other hand, nature has shown
numerous examples of evolutionary convergence where an enzyme catalyzing a highly specific,
O2-requiring reaction has an oxygen-independent counterpart, able to carry out the same reaction
under anoxic conditions. In this review, I attempt to take the reader on a simplified journey from
conventional nitrogenase complex to a possible simplified version of a yet to be discovered
light-utilizing nitrogenase (LUN).
Key words: aerobic, anaerobic, anoxic, convergent enzyme, light-independent, light-utilizing,
nitrogen fixation, nitrogenase, oxic, protochlorophyllide reductase
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Nitrogen was discovered by Daniel Rutherford in 1772. It was found to be so inert that Antoine
Lavoisier named it “azote”, meaning “without life”. Dinitrogen (N2) has a triple bond and does not
readily accept or donate electrons. As a gas or liquid, nitrogen is colorless and odorless. Two
allotropic forms of solid nitrogen exist, with the transition from the alpha to the beta form taking
place at -237°C. Like the global metabolism of many elements, the N cycle can be summarized as
transfer among the inorganic forms of nitrogen. Certain microorganisms have the ability to use the
renewable source of energy to fix atmospheric nitrogen (constituting 78% of air) under the mild
conditions, such as normal temperature and normal pressure. Nitrogen fixation is a key process in
which molecular nitrogen is reduced to form ammonia, which is the form of nitrogen that is used by
living systems for the synthesis of many bio-organic compounds. Biologically fixed nitrogen could
be directly ‘absorbed’ by plants and keep the environment almost ‘untouched’. Crop rotation with
legumes has been recognized to increase soil fertility and agricultural productivity since ancient
China and Rome. However, the science behind such practice was not revealed until Boussingault
experimented with leguminous crops fixing N2 in 1838; Hellriegel and Wilfarth showed definitive
evidence for N2 fixation by microbes in legumes in 1886. The Haber-Bosch industrial process was
established in 1906, which uses a catalytic agent (iron with a small amount of aluminum added) at
high pressure (as much as 500 atm) and high temperature (600-800 K) which normally consumes
fossil fuel. Annually, about 2.5 x 1011 kg NH 3 are fixed from atmosphere by biological nitrogen
fixation (by legumes and cyanobacteria) and about 8 x 1010 kg NH3 are manufactured by ammonia
industry. Lightning worldwide may also contribute about 1 x 1010 kg NH3 per year. Currently, about
two tons of industrially fixed nitrogen are needed as fertilizer for crop production to equal the
effects of one ton of nitrogen biologically fixed by legume crops. Therefore, biologically fixed
nitrogen influences the global nitrogen cycle substantially less than industrially fixed nitrogen. One
day, this situation needs to be changed.
On the other hand, world population has now been increasingly relying on nitrogen fertilizers in
order to keep up the demand of food and economic growth rates. As one can see, a large proportion
of fertilizers come from ammonia industry. However, less than 30% of synthetic fertilizers would
actually be utilized, the unused chemicals sprayed on crops would be lost in the field and could
subsequently cause serious environmental problems, let alone industrial pollution. Biological
nitrogen fixation has the advantage of being environmental friendly and therefore would be ideal
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for sustainable agriculture. Research in this field has pivotal importance and would be significantly
beneficial. Enormous progress in almost all aspects of biological nitrogen fixation has been made in
the past century, especially in the recent two decades, in genetics and biochemistry, culminating in
the determination of the crystallographic structures of both nitrogenase components. However, yet
more dynamic studies need to be carried out by biochemists, chemists, biophycists,
crystallographers, theoreticians, and genetists in order to completely understand the nature of the
process and make possible use of it.
3
Symbiotic model of legume-rhizobia
It has been discovered that at least three nitrogen fixation symbioses exist in nature: (1)
legume-rhizobia; (2) a range of species from fungi through ferns and cycads with cyanobacteria; (3)
non-leguminous angiosperms and the actinomycete Frankia spp. Legume-rhizobia symbiont is
most agriculturally important, and therefore mostly well studied. The current nodule formation
model is summarized in Figure 1. In initiation of the symbiosis, the rhizobia produce a
lipochitooligosaccharide-signaling molecule, Nod factor (NF), in response to rhizobia-derived
flavonoid molecules in the soil. The plant responds to NF within seconds, exhibiting ionic fluxes
and a calcium-spiking response in root hair cells (Mitra et al. 2004). The plant recognizes
rhizobia-signaling molecules and initiates transcriptional and developmental changes within the
root to allow rhizobia invasion and the construction of the nodule. The rhizobia reduce molecular
dinitrogen for use by the plant. Rhizobia nitrogenase is protected in an anaerobic environment by
the nodule cell wall and plant-producing leghemoglobin carries ‘excess’ oxygen away to where
respiration needs. FixABCX is responsible for electron transfer.
Figure 1. Schematic diagram of nodule formation. The host specificity is determined by two
methods in the mutual signal exchange between the plants and microsymbionts: Bacterial
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nodulation genes (nodABCD) are activated in response to plant-secreted signal molecules such as
flavonoids, resulting in biosynthesis and secretion of Nod factors, which elicit nodule formation on
the host plant (mainly legumes) roots and trigger the infection process.
In most plants ammonia is assimilated into amino acids through the cooperative activity of two
enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyzes the
incorporation of NH4+ into the amide position of glutamate, producing glutamine. GOGAT
catalyzes the reductive transfer of the amido group of glutamine to the α-keto position of
2-oxoglutarate, resulting in the formation of two molecules of glutamate.
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Assembly of the nitrogenase complex-- biochemistry and genetics
The most well-studied nitrogenase contains two metallo-components, dinitrogenase (MoFe protein)
and dinitrogenase reductase (Fe protein). The overall stoichiometry of dinitrogen reduction by
nitrogenase (EC 1.18.2.1) is: N2 + 8H+ + 8e- + 16MgATP
→ 2NH + H + 16MgADP + 16Pi
3
2
Nitrogenase turnover requires an electron donor in addition to ATP. Electrons are generated in vivo
either oxidatively or photosynthetically, depending on the organism. These electrons are transferred
to flavodoxin or ferredoxin, a [4Fe-4S]-containing electron carrier that transfers an electron to the
Fe protein of nitrogenase, beginning a series of oxido-reduction cycles. Two molecules of MgATP
bind to the reduced Fe protein and are hydrolyzed to drive an electron from the Fe protein to the
MoFe protein. The actual reduction of N2 occurs on the MoFe protein in a multi-step reaction.
Electron transfer must occur six times per each fixed N2 molecule so that a total of 12 ATPs are
required to fix one N2 molecule. However, nitrogenase also reduces protons to H2 a reaction which
consumes two electrons. Therefore, the total cost of N2 reduction is 8 electrons transferred and 16
MgATPs hydrolyzed (Figure 2).
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Figure 2. The mechanism of nitrogenase turnover. The reduction of N2 occurs on the MoFe
protein (α2β2 hetero-tetramer) in a multiple-step reaction with the Fe protein (γ2 homo-dimer).
Electron transfer six times per N2 molecule fixed and nitrogenase also reduces protons to H2,
consuming two electrons. The total cost of N 2 reduction is therefore 16 MgATPs hydrolyzed and 8
electrons transferred.
ATP hydrolysis, electron transfer and substrate reduction are the key steps for nitrogenase turnover.
The breakthrough of nitrogen fixation research was culminated by the structural characterization of
the nitrogenase components in late 20th century (Georgiadis et al. 1992; Kim et al. 1992, 1993;
Schindelin et al. 1997), which contributed significantly to our understanding of enzymatic nitrogen
reduction. The locations of the metal clusters in the nitrogenase components persuasively suggest a
general sequence of electron transfer: [4Fe4S]
→ [8Fe8/7S]→ [7Fe9S(Mo/V/Fe)]→ substrate
(Figure 3). The P-cluster [8Fe8/7S] is the electron “pool” which has almost equal distance (~14 Å)
7
to either Fe protein [4Fe4S] or FeMoco. The MoFe protein is an α2 β2 hetero-tetramer providing
substrate-binding and -reduction sites. The tetramer contains 30 Fe and 2 Mo atoms, which are
distributed between two types of cluster, the P-cluster [8Fe8S] in the Rees model or [8Fe7S] in the
Bolin model and the FeMo-cofactor (7Fe, 1 Mo, 9 S, 1 homocitrate) (Rees et al. 1993; Bolin et al.
1993). The α and β subunits are composed of 491 and 522 amino acids respectively in the case of
Av1, with a total molecular weight of about 240 KDa. The tetramer interface is dominated by
interactions between helices from the two β subunits, along with a cation binding site, presumably
occupied by calcium that is coordinated by residues from both β subunits. The contacts between the
αβ pairs are almost entirely between the β subunits. The P-cluster, which may function in electron
transfer between the [4Fe-4S] cluster and the FeMoco, is located about 10 Å beneath the protein
surface, on the 2-fold axis that approximately relates the α and β subunits. The FeMoco, which may
function in substrate binding and 6 reduction, is also buried about 10 Å beneath the protein surface
in an environment primarily provided by the α subunit. These amino acid environments not only
stablize the protein structure but also influence substrate reduction properties, such as the rate,
substrate specificity, and the products.
In the nitrogenase cycle, the role for ATP hydrolysis is to control the electron-transfer “gate”
between protein components. How this is accomplished is still one of the two main unanswered
questions about the nitrogenase mechanism (the other one being how substrates are reduced at the
FeMoco). There is excellent evidence that the FeMo-cofactor clusters act as the enzyme’s
substrate-binding and -reducing site, but exactly how and where substrates bind and are activated
remains controversial. A very close observation was reported with a high-resolution structure of the
MoFe protein of nitrogenase, which reveals a previously unrecognized interstitial atom in the
FeMo cofactor that may possibly be nitrogen (Einsle et al. 2002). Electron transfer from the Fe
protein to the MoFe protein is coupled to the hydrolysis of MgATP which is followed by
dissociation of the protein-protein complex. The electron transfer step is an essential function of the
Fe protein, since the MoFe protein alone will not reduce N2 in the absence of the Fe protein, despite
the fact that the MoFe protein component (cofactor) can be reduced by other electron donors.
8
Figure 3. Complex of the nitrogenase proteins (Fe protein and MoFe protein). The individual
subunits of each Fe protein colored green and yellow. The MoFe
- and -subunits colored red
and blue, respectively. Non-protein groups are shown in a space-filling representation, with
fluorine and magnesium colored orange and green, respectively. Transduction pathway coupling
the nucleotide and cofactor sites in the nitrogenase complex: [4Fe4S]-cluster, P-cluster, and
FeMo-cofactor sites (Howard and Rees, 2006).
In nitrogen fixing bacteria, nitrogenase is encoded by a set of operons which includes regulatory
genes (such as nifLA), structural genes (such as nifHDK) and other supplementary genes. The
free-living diazotrophic bacterium Klebsiella pneumoniae has been the most extensively analysed
and provides a model for studies of nitrogenase regulation, synthesis and assembly. A 24kb base
pair DNA region, contains the entire K. pneumoniae nif cluster, which includes 20 genes (Figure 4).
nifHDK are the three structural genes encoding for the three subunits of Mo-nitrogenase. In most
nitrogen fixing prokaryotes, these three genes form one transcriptional unit, with a promoter in
front of the nifH gene. The maturation of apo-Fe protein (NifH) requires the products of nifH, nifM,
nifU and nifS, while that of apo-MoFe protein requires at least 6 genes nifE, nifN, nifV, nifH, nifQ
and nifB which are required for the biosynthesis of FeMoco. There is considerable homology
between nifDK and nifEN, and it has been speculated that the nifEN products might form a scaffold
for FeMoco biosynthesis that later shifts FeMoco to the nifDK complex. The nifB gene product,
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termed NifB-co, is an iron- and sulfur-containing precusor of FeMoco. The nifQ gene product
might be involved in the formation of a molybdenum-sulfur precursor to FeMoco and it has a
typical motif characteristic of metal-binding sequences (Cys-X4-Cys-X2-Cys-X5-Cys). The nifV
gene encodes a homocitrate synthase and is required for the synthesis of FeMoco. nifW is not
required for the initial assembly of the MoFe protein but rather may be necessary to protect the
MoFe protein from O2 damage. The nifY gene product has a function similar to gamma protein.
nifF and nifJ encode components of a specific electron transfer pathway in which electrons are
donated from pyruvate to a flavodoxin and hence to the Fe protein of nitrogenase. Although nifM
has been found necessary for the maturation and stablization of the nifH product, its actual role has
never been defined. In contrast, nifS and nifU are ubiquity in various organisms (Dos Santos et al.
2004).
Figure 4. The K. pneumoniae nif cluster including 20 genes (~24 kb). Transcriptional
orientation, protein product molecular weights and known functions are indicated.
10
Complexity of nitrogenase regulation-- buttons of switch on/off
Nitrogenase is highly regulated in vivo largely due to nitrogen fixation being a highly energy
consuming and oxygen sensitive process. Therefore, biosynthesis of nitrogenase stringently senses
the redox/nitrogen status via several regulatory protein systems in various bacteria. NifL, NifA,
FixL, FixJ, GlnD, GlnB (PII), NtrB, NtrC, NtrA (RpoN,
σ , RNA polymerase sigma factor) and
N
RegB (PrrB, RegS or ActS) are such regulatory proteins.
In the Proteobacteria nif genes are invariably subject to transcriptional activation by NifA, a
member of the EBP (enhancer-binding protein) family, together with the RNA polymerase sigma
factor, σ54. σ54 reversibly associates with the core RNA polymerase to recognize promoters with
consensus sequences at –12 and –24 bp relative to the transcription start site and transcription
initiation is dependent on interaction with a member of the EBP family. NifA is the master regulator
of nitrogen fixation and, although regulatory cascades differ, each regulatory circuit ultimately
results in regulation of NifA expression or modulation of its activity in response to oxygen and/or
fixed nitrogen. NifA has a domain architecture that is similar to other members of the EBP family
in which a central, conserved AAA+ ATPase domain is flanked by an amino-terminal regulatory
domain and a carboxy-terminal DNA-binding domain, which contains a helix–turn–helix motif that
is required for recognition of the UAS enhancer-like elements. Interaction of the EBP with the σ54
RNA polymerase holoenzyme is facilitated by the binding of the activator to DNA sequences
(upstream activator sequences, UAS) usually located at least 100 bp upstream of the transcription
initiation site. DNA looping is required to establish productive interactions between the
DNA-bound activator and the polymerase. In some cases this is assisted by other DNA-binding
proteins, such as IHF (integration host factor) (Figure 5). In symbiotic diazotrophs, transcription of
nifA and fix genes is predominantly controlled by the oxygen-responsive two component
FixL–FixJ system. In addition to regulation at the transcriptional level, nitrogenase activity is also
subject to post-translational regulation in some diazotrophs — for example in Rhodospirillum
rubrum, Rhodobacter
capsulatus and
Azospirillum
brasilense. This is mediated by
ADPribosylation of the Fe protein of nitrogenase by DraT (dinitrogenase reductase
ADP-ribosyltransferase), which inactivates nitrogenase in response to ammonium and light
intensity. Covalent modification of nitrogenase is reversed by DraG (dinitrogenase reductase
activating glycohydrolase), which removes the ADP–ribose moiety under conditions that are
11
appropriate for nitrogen fixation (Halbleib et al. 2000).
Figure 5. Enhancer-binding proteins working model. (a) Enhancer-binding proteins (EBPs) are
a unique class of prokaryotic transcriptional activators that interact with the RNA polymerase
sigma factor, σ54. σ54 reversibly associates with the core RNA polymerase to recognize promoters
with consensus sequences at –12 and –24 bp relative to the transcription start site and transcription
initiation is dependent on interaction with a member of the EBP family; (b) Interaction of the EBP
12
with the σ54 RNA polymerase holoenzyme is facilitated by the binding of the activator (shown as a
blue oligomer) to DNA sequences (upstream activator sequences, UAS) usually located at least 100
bp upstream of the transcription initiation site. DNA looping is required to establish productive
interactions between the DNA-bound activator and the polymerase. In some cases this is assisted
by other DNA-binding proteins, such as integration host factor (IHF); (c) In the absence of the EBP,
σ54 RNA polymerase holoenzyme forms closed promoter complexes (in which the promoter DNA
is doublestranded) that rarely undergo spontaneous isomerization to form open complexes (in
which the DNA strands surrounding the transcription start site are locally denatured). Nucleotide
hydrolysis by the activator promotes remodelling of the closed complex through a series of
protein–protein and protein–DNA interactions that favour conversion to the open promoter
complex; (d) EBPs have a modular domain structure, which frequently comprises at least three
domains: one or more amino-terminal regulatory domain (which often belong to the GAF, PAS or
response-regulator families), a highly conserved central domain that is required for the
nucleotide-dependent interactions that drive open complex formation by σ54 RNA polymerase, and
a carboxy-terminal DNA-binding domain (Dixon and Kahn 2004). Figure reproduced with
permission © (2004) nature publishing group.
Both A. vinelandii and K. pneumoniae have similar NifL–NifA systems in which NifL controls
NifA activity in response to the nitrogen source. However, the mechanisms by which this is
achieved in conjunction with PII-like signal-transduction proteins are very different. Like many
other Proteobacteria, A. vinelandii encodes proteins that are required for general nitrogen control,
including a PII-like protein, uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and the
NtrB–NtrC two component regulatory system (Figure 6).
13
Figure 6. The ntr system composed of four enzymes: a uridyltransferase/uridylyl-removing
enzyme (UTase/UR), encoded by the glnD gene; a small trimeric protein, P II, encoded by glnB; a
two-component regulatory system composed of the histidine protein kinase NtrB and the response
regulator NtrC. The phosphorylated form of NtrC is needed for transcription of the regulatory nifLA
operon, NtrB controls the phosphorylation state of NtrC. Phosphorylation of NtrC requires NtrB
and the uridylylated form of PII. Uridylylation of PII is regulated by GlnD, with nitrogen limitation
favoring uridylylation and nitrogen excess conditions favoring removal of the uridylyl residue.
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Nitrogenase has ‘siblings’-- double-bond reductases for chlorophyll biosynthesis in
photosynthesis
Chlorophyll is essential for life in the biosphere, playing an important role in the energy absorption
and transduction processes of photosynthetic organisms. Chlorophyll catalyzes the conversion of
solar energy to chemical energy via the process of photosynthesis. Approximately 250-300 of them
transfer the absorbed light energy through neighboring pigments to the “special pair” of
chlorophylls in a reaction center. This special pair of chlorophylls in photosystems I and II are the
primary electron donors that drive the conversion of light into chemical energy to be conserved in
NADPH and ATP. The most important pigment molecule in photosynthesis is chlorophyll a, which
absorbs light only at certain wavelengths. The process of photosynthesis can capture more energy if
it uses other molecules, accessory pigments, to absorb the energy from other wavelengths and pass
it on to chlorophyll a. The reduction of Pchlide is a key step in this biosynthesis of Chl during the
greening of phototrophic organisms. The process is catalysed by the key enzyme
NADPH::protochlorophyllide oxidoreductase (LPOR; EC 1.3.1.33). With the exception of
angiosperms, a light-independent chlorophyll biosynthesis pathway has been found and catalyzed
by the light-independent enzyme DPOR (dark-operative protochlorophyllide oxidoreductase) at the
key step of converting protochlorophyllide to chlorophyllide a (Figure 7).
Figure 7. Chlorophyll biosynthesis pathway. Protochlorophyllide reductase catalyzing
protochlorophyllide to chlorophyllide a by DPOR (exist up to gymnosperm ) or LPOR (dominant
in angiosperm).
15
motif A
NIFH_AZOCH
MAMRQCAIYGKGGIGKSTTTQNLVAALAEMGKKVMIVGCDPKADSTRLILHSKAQNTIME 60
NIFH_AZOVI
MAMRQCAIYGKGGIGKSTTTQNLVAALAEMGKKVMIVGCDPKADSTRLILHSKAQNTIME 59
NIFH_KLEPN
-TMRQCAIYGKGGIGKSTTTQNLVAALAEMGKKVMIVGCDPKADSTRLILHAKAQNTIME 59
CHLL_CHLRE
---MKLAVYGKGGIGKSTTSCNISIALRKRGKKVLQIGCDPKHDSTFTLTGFLIPTIIDT 57
: *:***********: *: ** : ****: :***** *** :
. *
NIFH_AZOCH
MAAEAGTVEDLELEDVLKVGYGGVKCVESGGPEPGVGCAGRGVITAINFLEEEGAYEDDL 120
NIFH_AZOVI
MAAEAGTVEDLELEDVLKAGYGGVKCVESGGPEPGVGCAGRGVITAINFLEEEGAYEDDL 119
NIFH_KLEPN
MAAEVGSVEDLELEDVLQIGYGDVRCAESGGPEPGVGCAGRGVITAINFLEEEGAYEDDL 119
LSSKDYHYEDIWPEDVIYGGYGGVDCVEAGGPPAGAGCGGYVVGETVKLLKELNAFF-EY 116
CHLL_CHLRE
::::
**: ***: ***.* *.*:*** .*.**.* * ::::*:* .*: :
motif B
NIFH_AZOCH
DFVFYDVLGDVVCGGFAMPIRENKAQEIYIVCSGEMMAMYAANNISKGIVKYANSGSVRL 180
NIFH_AZOVI
DFVFYDVLGDVVCGGFAMPIRENKAQEIYIVCSGEMMAMYAANNISKGIVKYANSGSVRL 179
NIFH_KLEPN
DFVFYDVLGDVVCGGFAMPIRENKAQEIYIVCSGEMMAMYAANNISKGIVKYAKSGKVRL 179
CHLL_CHLRE
DVILFDVLGDVVCGGFAAPLN--YADYCIIVTDNGFDALFAANRIAASVREKARTHPLRL 174
*.:::************ *:.
*:
** .. : *::***.*: .: : *.: :**
NIFH_AZOCH
GGLICNSRNTDREDELIIALAAKLGTQMIHFVPRDNVVQRAEIRRMTVIEYDPTAKQADE 240
NIFH_AZOVI
GGLICNSRNTDREDELIIALANKLGTQMIHFVPRDNVVQRAEIRRMTVIEYDPKAKQADE 239
NIFH_KLEPN
GGLICNSRQTDREDELIIALAEKLGTQMIHFVPRDNIVQRAEIRRMTVIEYDPACKQANE 239
CHLL_CHLRE
AGLIGN-RTSKRD--LIDKYVEACPMPVLEVLPLIEEIRISRVKGKTLFEMSNKNNMTSA 231
.*** * * :.*: **
.
::..:* : :: :.:: *::* .
: :.
NIFH_AZOCH
YRTLARKVVENKMLIIPNPITMDELEALLMEFGVMEEEDESIVGKAAAAEE--------- 291
NIFH_AZOVI
YRALARKVVDNKLLVIPNPITMDELEELLMEFGIMEVEDESIVGKTAEEV---------- 289
NIFH_KLEPN
YRTLAQKIVNNTMKVVPTPCTMDELESLLMEFGIMEEEDTSIIGKTAAEENAA------- 292
CHLL_CHLRE
HMDGS-KGDNSTVGVSETPSEDYICNFYLNIADQLLTEPEGVIPRELADKELFTLLSDFY 290
:
: * :..: : .*
NIFH_AZOCH
---
NIFH_AZOVI
---
NIFH_KLEPN
---
CHLL_CHLRE
LKI 293
: *
. : * .:: :
Figure 8. Amino acid sequence alignment between ChlL and NifH. Similar nucleotide binding motif A
(GXXXXGK15S), motif B (D125XXG) and conserved cysteines for liganding the [4Fe-4S] cluster (Cys97 and Cys132)
are found. The identical residues are indicated by asterisks. CHL_CHLRE: Chlamydomonas Reinhardtii ChlL;
NIFH_KLEPN: Klebsiella pneumoniae NifH; NIFH_AZOCH: Azotobacter chroococcum NifH; NIFH_AZOVI:
Azotobacter vinelandii NifH.
16
Genes for such light-independent pathways of chlorophyll biosynthesis were first discovered when
the complete chloroplast genome sequence from the liverwort Marchantia polymorpha was
determined (Ohyama et al., 1986). A gene designated frxC (later renamed as chlL) at that time was
found to have a high degree of similarity with nifH of nitrogenase (Figure 8). Genetic studies in
Rhodobacter capsulatus also demonstrated that three genes, bchL, bchN, and bchB, were involved
in protochlorophyllide reduction (Zsebo and Hearst 1984; Bauer et al. 1988; Yang and Bauer 1990).
Sequence analysis revealed the surprising finding that these genes, together with genes identified
from cyanobacterium, algae and germnosperms, termed chlL, chlN, and chlB exhibit striking
similarities to the three subunits of nitrogenase (Lidholm and Gustafsson 1991; Fujita et al. 1992,
1993; Li et al. 1993; Liu et al. 1993). This suggests that light-independent protochlorophyllide
reductase and nitrogenase share a common evolutionary ancestor (Burke et al. 1993; Fujita 1996).
In nitrogenase, the Fe protein (structurally encoded by nifH) specifically transfers electrons to the
MoFe protein (structurally encoded by nifD and nifK) in a reaction that is coupled with the
hydrolysis of MgATP (see the nitrogenase mechanism). ChlL and ChlB are similar to NifH (about
35%) and NifK (about 19%), respectively (Fujita et al., 1991). ChlN is similar to both NifD and
NifK (about 19%) (Fujita et al. 1993) and in particular, has cysteine residues at positions equivalent
to Cys α62, α88, α154, β70, β52, β95, β153, which coordinate the P clusters in nitrogenase MoFe
protein. In addition, some similarity (about 16%) between ChlN and ChlB is also apparent, as it is
between NifD and NifK (Fujita et al. 1996). Similarities between the subunits of DPOR and those
of nitrogenase suggest that DPOR has a molecular architecture similar to that of nitrogenase
(Figure 9). It’s been increasingly evident that in the DPOR complex, ChlL contains a [4Fe-4S]
cluster, functioning as a specific donor of electrons to the other component, consisting of ChlN and
ChlB, which catalyze the reduction of Pchlide (Fujita and Bauer 2000; Cheng et al. 2005a;
(Brocker et al. 2008). In the synthesis of Bchl, the reduction of the chlorin B-ring double-bond in
chlorophyllide a (λmax: 661 nm) to bacteriochlorophyllide a (λmax: 771 nm) is sequentially
catalyzed by chlorophyllide reductase (COR) (Nomata et al. 2006). COR is encoded by bchX, bchY
and bchZ, which are also homologous to chlL, chlN and chlB, respectively (Burke-Aquero 1992;
Burke et al. 1993). The similarities among DPOR, COR and nitrogenase suggest an evolutionary
relationship. It appears that nitrogenase may have more than one ‘sibling’.
17
Figure 9. DPOR structural model and comparison with nitrogenase. Electron transfer pathway
→ ChlNB [4Fe4S]-center → ChlNB [4Fe4S]-center versus in
nitrogenase: NifH [4Fe4S]-cluster → NifDK [P-cluster] → NifDK [FeMo-cofactor].
in DPOR: ChlL [4Fe4S]-cluster
Of great interest, from the protein point of view, is that nitrogenase remains ‘prokaryotic’ while its
‘sibling’ (DPOR) evolves into a higher version (LPOR), ultimately becoming dominate in higher
plants (angiosperms). DPOR and LPOR are very different proteins and structurally unrelated, but
carry out similar reactions with different chemistry (Figure 10). LPOR occurs in all oxygenic
photosynthetic organisms. With the exception of angiosperms, LPOR coexists with DPOR,
although DPOR is not essential and may not become redundant. LPOR is apparently absent from
anoxygenic bacteria, which have only DPOR enzymes (Suzuki and Bauer 1995; Schoefs and
Franck 2003). LPOR gene originated in the cyanobacterial genome before the divergence of oxic
photosynthetic organisms. The photosynthetic eukaryotes obtained their LPOR homologues
through endosymbiotic gene transfer (Yang and Cheng 2004). In green algae and higher plants,
LPOR is encoded in the nucleus, translated as a precursor polypeptide in the cytosol, and ultimately
post-translationally processed and imported into plastids. Comparison of the primary and
18
secondary structure of LPORs with other proteins requiring NADPH as co-factor has revealed that
LPOR belongs to an extended superfamily of NAD(P)H-accepting oxidoreductase, termed the
short-chain dehydrogenase/reductase (SDR) family (Figure 11). A most striking feature of LPOR is
that it requires light for its catalytic activity, one of the only two photo-enzymes (Dahlin et al.
1999).
Figure 10. DPOR and LPOR are different proteins, but carrying out similar reactions with
different chemistry.
19
Figure 11. Neighbor joining tree showing the relationships between LPORs and other
classical SDRs. The species are included in Archaea, Purple bacteria, green bacteria and
cyanobacteria (Yang and Cheng, 2004).
20
Efforts on introduction of nitrogenase gene into chloroplast genome in non-legume plant
More than three decades ago when the nif genes were successfully transferred into Escherichia coli
from the nitrogen fixing bacterium K. pneumoniae (Dixon and Postgate 1972) the transfer of nif
genes directly into plant cell to create diazotrophic plants has also been considered (Earl and
Ausubel 1982). Several possible locations for expression of nitrogenase in the plant cell have been
suggested (Merrick and Dixon 1984) and attempts to express nif genes in higher plants was
achieved by targeting NifH or NifH plus NifM into tobacco chloroplasts, although in both cases
NifH was found in the chloroplast stromal fraction at a very low level (Dowson-Day et al. 1991).
There are advantages for the chloroplast being a potential environment for nitrogen fixation: the
developed plastid carries out photosynthesis and thus provides a major source of ATP. The
chloroplast is also a major site for ammonia assimilation since both the GS and the GOGAT
pathways exist in the chloroplast. From an engineering point of view, the plastid provides a
convenient location for the introduction of nif genes since chloroplast genes are expressed in a
prokaryotic-like fashion, allowing translation of polycistronic messages. Chlamydomonas
possesses a light-independent pathway for chlorophyll biosynthesis, with one of the enzymes in the
pathway having potential structural and functional homologies to nitrogenase. The products of the
C. reinhardtii chlL, N and B genes are structurally similar to the three subunits of nitrogenase, with
the strongest sequence identity between nifH and chlL (nucleotide sequence 43%; putative amino
acid sequence 33%) (Figure 8). Therefore, the genes required for chlL protein activity might
activate the nifH gene product to obtain Fe protein activity without the requirement for additional
genes such as nifM, nifS, or nifU. Although regulation of the chlL, N and B genes is not yet clear,
using the native chl system may provide a strategy for the expression of nifH in an active form. In
addition, the nifH gene product might substitute for the function of chlL. The first step was to
precisely replace the coding region of chlL gene with that of nifH gene, keeping the untranslated
regulatory regions intact. We designed a strategy to introduce nifH into the chloroplast genome by
firstly creating a petB::aadA insertion mutation and subsequently converted it back to wild-type
petB with a second homologous recombination event which introduces nifH. This event places nifH
under the control of flanking chlL5’ and 3’ regulatory regions (Figure 12). Our results demonstrate
that the nifH gene product does substitute for the function of chlL and it is possible that the
chloroplast contains similar ancillary proteins for the biosynthesis of [4Fe-4S] proteins (Cheng et
21
al., 2005a).
Figure 12. Schematic diagrams of constructs and two-step chloroplast transformation,
introduction of nitrogenase Fe protein (NifH) into chloroplast genome of Chlamydomonas
reinhardtii by replacing its native ChlL. (A) (a) Chloroplast transformation vector pCQ3. The
aadA cassette is inserted in the opposite orientation in the petB coding region; (b) expression vector
pCQ5, containing multiple-cloning-site between chlL 5’ and 3’ untranslated regulatory sequences
for insertion of foreign target genes; (c) secondary chloroplast transformation vector pCQ7,
containing uidA gene driven by chlL promoter; (d) secondary chloroplast transformation vector
pCQ9, containing nifH gene driven by chlL promoter. (B) Two-step chloroplast transformation via
homologous recombination by bombardment vector pCQ3 to obtain petB mutant which was used
as a recipient for the secondary transformation by delivering vector pCQ9 bearing nifH gene to
22
obtain C. reinhardtii nifH transplastomic line. C. reinhardtii uidA transplastomic line was also
achieved by this strategy (Cheng et al., 2005a).
One of the next possible approaches would be the replacement of the nifDK-like DPOR component
ChlN and ChlB genes with that of Mo-nitrogenase structural nifDK genes. Based on the similar
assumption that the ChlNB complex may resemble the NifDK complex harboring a similar metal
scaffold, provided by yet unknown DPOR biogenesis proteins in chloroplasts, the expectation is to
alter the enzymatic DPOR structure towards a functional nitrogenase in vivo.
However, it is a major challenge to attempt to engineer a nitrogen-fixing organelle, we still have to
face the problem of interfacing plastid physiology with the requirements for nitrogenase activity.
As an alternative strategy, one might consider introducing nitrogenase proteins into mitochondria
which may provide a suitable energy-rich, reducing environment to support nitrogenase. However,
this introduces more complexity since each gene would have to be specifically modified to allow
targeting each of the nif-encoded proteins into this organelle in the appropriate stoichiometry.
Paradoxically, whilst the technology is available now to introduce and express nitrogenase
component proteins in plant cells, substantial progress is limited by gaps in our fundamental
knowledge of both plant and microbial physiology.
23
The emergence of non-conventional nitrogenase and further predictions
Like everything else on our living planet earth, nitrogenase has been evolving in a dynamic way.
Alternative nitrogenases discovered in Azotobacter vinelandii using vanadium, iron, instead of
molybdenum in an environment lacking molybdenum metal. Both conventional nitrogenase and
alternative nitrogenase are two-component complexes and both components are highly sensitive to
O2. Streptomyces thermoautotrophicus is recently found to be able to fix dinitrogen, but it harbors a
very unusual N2-fixing system that requires three proteins for nitrogen fixation, a heterotrimeric
molybdenum-containing dinitrogenase (St1), a homodimeric manganese-containing superoxide
oxidoreductase (St2) and another heterotrimeric molybdenum-containing carbon monoxide
dehydrogenase (St3 or CODH) (Figure 13). These proteins differ entirely from the known
nitrogenase protein components and show insensitiveness to O 2. Compared to conventional or
alternative nitrogenases, the St nitrogenase also requires less ATP: N2 + 8H+ + 8e- + (4-12)MgATP
→ 2NH3 + H2 + (4-12)MgADP + (4-12)Pi
Figure 13. Schematic representation of N2 fixation in S. thermoautotrophicus (Figure
reproduced with permission). Superoxide is produced by CO dehydrogenase through the oxidation
of CO and the transfer of the electrons to O2. Subsequently, the superoxide is reoxidized by a
superoxide oxidoreductase that delivers the electrons to a dinitrogenase. The dinitrogenase is
capable of reducing N2 and H+, but not ethane (Ribbe, M. et al. 1997).
Previously, S. thermoautotrophicus UBT1 was isolated from burning charcoal pile. The N2-fixing
ability of S. thermoautotrophicus was discovered by growing the strain chemolithoautotrophicaily
24
with CO or H2 plus CO2 under aerobic conditions at 65°C (Gadkari et al. 1992; Ribbe, M. et al.
1997). That is indeed a good ‘selection’ system where N2 would be the sole nitrogen source.
Another striking characteristic of St nitrogenase is that it is not inhibited by CO, which is the case
for the conventional nitrogenase. So far, all known nitrogenases are found in complex prokaryotic
versions. There may be more types of prokaryotic nitrogenases with versatile features to be
discovered. Since many prokaryotic enzymes do evolve into eukaryotic version, it would be
difficult to rule out the possibility of the existence of a eukaryotic nitrogenase (Yang and Cheng,
2004); taking one step further, if eukaryotic nitrogenase does exist in nature, then it may well be
utilizing light as energy source (Cheng et al., 2005b) [LPOR-like nitrogenase: Light-utilizing n2ase
(LUN)] (Figure14). The natural history of DPOR and LPOR is a perfect example and indication,
driving research to reveal the possible existance of biological nitrogen fixation in wild species of
non-legume plants, i.e. possible existence of another non-conventional nitrogenase in plant (Figure
14, 15).
Figure 14. Possibility of the existence of non-conventional nitrogenase. Conventional
nitrogenase complex convert N2 into NH3 by using ATP as energy; if a LPOR-like enzyme or
non-LPOR enzyme could do the same catalysis by a single polypeptide, it could well be a
light-utilizing nitrogenase (LUN).
25
One possibility may be as suggested in Figure 15 showing relative appearances of nitrogenase,
DPOR, LPOR and putative nitrogenase in geologic time (past and future). Such predictions are
encouraged by the fact that the anaerobic enzyme has almost certainly been replaced in many
reactions by the more efficient and irreversible aerobic version. On the other hand, nature has
shown several examples of evolutionary convergence where an enzyme catalyzing a highly specific,
O2-requiring reaction has an oxygen-independent counterpart, able to carry out the same reaction
under anoxic conditions (Raymond and Blankenship 2004). While many unpublished experiments
have been conducted to achieve perfect CO2/O2 ratio by engineering Rubisco small subunits
(Spreitzer et al. 2005), a better Rubisco enzyme has been found already existing in nature
(personnal communications). Nevertheless, efforts need to be made for an extensive investigation
with an open mind and rational searching/selection system, towards finding more enzymes that are
able to achieve N2-fixing for microbes or even a eukaryotic version for plants. Hundreds of genome
projects have now been completed, leaving on average more than 50% of discovered proteins
waiting to be assigned functions. The speed of genome-wide data accumulation is exponential.
Regarding the possible origin of LUN, might it be the same as LPOR as from SDR protein family,
might it be one of the LPOR, or from a completely different family, or never even have existed at
all. Perhaps no plant is able to fix nitrogen? Do plants really not fix nitrogen without a nodule? So
far, a definite answer is difficult. On the other hand, if such proteins do exist, a network might
already be in place, saving people from having to undergo sophisticated ‘metabolic engineering’; if
not, one may eventually figure out the scientific reason behind it. Either way it may certainly press
science forward in 21st century.
At this point, one may naively begin investigating the idea of massive over-expression of such
proteins heterologously in bacteria. One may never find it active in the sense that current popular
methods for assaying nitrogenase are based on C2H2 reduction and H2 evolution. Bearing in mind,
to achieve today’s knowledge about microbial N2-fixing, it took over a hundred years of research in
the field of biological nitrogen fixation concentrating mostly on symbiosis models. However, the
speed of discovery in modern science should never be underestimated.
Just as B. E. Smith once stated “history tells us that, in this field, the one prediction that we can
safely make is that there will be surprises awaiting us”.
26
Figure 15. A schematic diagram high-lighting the origin of N2ase, DPOR, LPOR and
putative LUN. O- origin of our planet earth, formed approximately 4~4.5 Gya ago; E- life of the
earth (it’s been estimated that it could live on for at least another 4~5 Gya); n- any geologic time in
the future when some old fashion enzymatic systems might be completely wiped out by nature;
N2ase appeared at very early stage of an anaerobic world; DPOR was evolved from the same
ancestor as n2ase; LPOR appeared when there was considerable O2 in the atmosphere; LUN1 could
have existed around the same period of the origin of LPOR or there may be a possible later
evolutionary event giving birth to LUN2 by an unknown driving force in nature or lab.
27
Acknowledgements
I thank R. Dixon for the diagram used in Figure 5, and many of my colleagues for helpful
discussions and apologize for not thanking them individually. This review is dedicated to C. B. You,
who sadly passed away in 1994, and will be remembered as one of the early researchers in the field
of biological nitrogen fixation in China.
28
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